Methods of modulating immune responses using bcma polypeptide

ABSTRACT

A novel receptor in the TNF family is provided: BAFF-R. Chimeric molecules and antibodies to BAFF-R and methods of use thereof are also provided.

RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 15/595,733,which is a divisional of U.S. patent application Ser. No. 14/452,870,filed Aug. 6, 2014 (now U.S. Pat. No. 9,650,430, issued May 16, 2017),which is a continuation of U.S. patent application Ser. No. 13/401,610,filed Feb. 21, 2012 (now U.S. Pat. No. 8,828,669, issued Sep. 9, 2014),which is a continuation of U.S. patent application Ser. No. 12/500,909,filed Jul. 10, 2009 (abandoned), which is a continuation of U.S. patentapplication Ser. No. 10/077,137, filed Feb. 15, 2002 (now U.S. Pat. No.7,691,804, issued Apr. 6, 2010), which is a continuation ofInternational Application No. PCT/US00/22507, filed Aug. 16, 2000, whichclaims priority from U.S. Patent Application No. 60/149,378, filed Aug.17, 1999, U.S. Patent Application No. 60/181,684, filed Feb. 11, 2000,and U.S. Patent Application No. 60/183,536, filed Feb. 18, 2000, each ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the use of a receptor to BAFF, a β-cellactivating factor belonging to the Tumor Necrosis Factor (“TNF”) family,and its blocking agents to either stimulate or inhibit the expression ofB-cells and immunoglobulins. This receptor has anti-cancer andimmunoregulatory applications as well as uses for the treatment ofimmunosuppressive disorders such as HIV. In addition, the receptor andits blocking agents play a role in the development of hypertension andits related disorders. Furthermore, cells transfected with the gene forthis receptor may be used in gene therapy to treat tumors, lymphomas,autoimmune diseases or inherited genetic disorders involving B-cells.Blocking agents, such as recombinant variants or antibodies specific tothe receptor, have immunoregulatory applications as well. Use of thereceptor to BAFF as a B-cell stimulator for immune suppressed diseasesincluding for example uses for patients undergoing organ transplantation(e.g., bone marrow transplant) as well as recovering from cancertreatments to stimulate production of B cells are contemplated. Use ofthe receptor to BAFF as an adjuvant and or costimulator to boost and/orrestore B cell levels to approximately normal levels are alsocontemplated. Soluble forms of the receptor to BAFF that block B cellfunction may also be used to inhibit B-cell mediated diseases.

BACKGROUND OF THE INVENTION

The present invention relates to a novel receptor in the TNF family. Anovel receptor has been identified, BAFF-R (or “BCMA”).

The TNF family consists of pairs of ligands and their specific receptorsreferred to as TNF family ligands and TNF family receptors (Bazzoni andBeutler, 1996). The family is involved in the regulation of the immunesystem and possibly other non-immunological systems. The regulation isoften at a “master switch” level such that TNF family signaling canresult in a large number of subsequent events best typified by TNF. TNFcan initiate the general protective inflammatory response of an organismto foreign invasion that involves the altered display of adhesionmolecules involved in cell trafficking, chemokine production to drivespecific cells into specific compartments, and the priming of variouseffector cells. As such, the regulation of these pathways has clinicalpotential.

Induction of various cellular responses mediated by such TNF familycytokines is believed to be initiated by their binding to specific cellreceptors. At least two distinct TNF receptors of approximately 55 kDa(TNFR1) and 75 kDa (TNFR2) have been identified [Hohman et al., J. Biol.Chem. 264: 14927-14934 (1989) and Brockhaus et al., PNAS, 87: 3127-3131(1990)]. Extensive polymorphisms have been associated with both TNFreceptor genes. Both TNFRs share the typical structure of cell surfacereceptors including extracellular, transmembrane and intracellulardomains. The extracellular portion of type 1 and type 2 TNFRs contains arepetitive amino acid sequence pattern of four cysteine rich domains(CDRs). A similar repetitive pattern of CDRs exist in several other cellsurface proteins, including p75 nerve growth factor receptor, the B-cellantigen CD40 amongst others.

The receptors are powerful tools to elucidate biological pathwaysbecause of their easy conversion to immunoglobulin fusion proteins.These dimeric soluble receptor forms are good inhibitors of eventsmediated by either secreted or surface bound ligands. By binding tothese ligands they prevent the ligand from interacting with cellassociated receptors that can signal. Not only are these receptor-Igfusion proteins useful in an experimental sense, but they have beensuccessfully used clinically in the case of TNF-R-Ig to treatinflammatory bowel disease, rheumatoid arthritis and the acute clinicalsyndrome accompanying OKT3 administration (Eason et al., 1996; Feldmannet al., 1996; van Dullemen et al., 1995). One can envision thatmanipulation of the many events mediated by signaling through the TNFfamily of receptors will have wide application in the treatment ofimmune based diseases and also the wide range of human diseases thathave pathological sequelae due to immune system involvement. A solubleform of a recently described receptor, osteoprotegerin, can block theloss of bone mass and, therefore, the events controlled by TNF familyreceptor signaling are not necessarily limited to immune systemregulation. Antibodies to the receptor can block ligand binding andhence can also have clinical application. Such antibodies are often verylong-lived and may have advantages over soluble receptor-Ig fusionproteins which have shorter blood half-lives.

While inhibition of the receptor mediated pathway represents the mostexploited therapeutic application of these receptors, originally it wasthe activation of the TNF receptors that showed clinical promise(Aggarwal and Natarajan, 1996). Activation of the TNF receptors caninitiate cell death in the target cell and hence the application totumors was and still is attractive (Eggermont et al., 1996). Thereceptor can be activated either by administration of the ligand, i.e.the natural pathway or some antibodies that can crosslink the receptorare also potent agonists. Antibodies would have an advantage in oncologysince they can persist in the blood for long periods whereas the ligandsgenerally have short lifespans in the blood. As many of these receptorsmay be expressed more selectively in tumors or they may only signal celldeath or differentiation in tumors, agonist antibodies could be goodweapons in the treatment of cancer. Likewise, many positiveimmunological events are mediated via the TNF family receptors, e.g.host inflammatory reactions, antibody production etc. and thereforeagonistic antibodies could have beneficial effects in other,non-oncological applications.

Paradoxically, the inhibition of a pathway may have clinical benefit inthe treatment of tumors. For example the Fas ligand is expressed by sometumors and this expression can lead to the death of Fas positivelymphocytes thus facilitating the ability of the tumor to evade theimmune system. In this case, inhibition of the Fas system could thenallow the immune system to react to the tumor in other ways now thataccess is possible (Green and Ware, 1997).

SUMMARY OF THE INVENTION

Applicants have identified a cDNA clone that encodes a polypeptide,designated in the present application as “BAFF-R” or as “BCMA”, thatbinds the tumor necrosis factor, BAFF (SEQ ID NO: 9), a B-cellactivating factor belonging to the Tumor Necrosis Factor (“TNF”) family.BAFF is the same molecule previously described in WO/9912964, which isincorporated by reference herein.

In one embodiment, the invention provides methods of using BAFF-R.Included in such methods are methods of inhibiting B-cell growth,dendritic cell-induced B-cell growth and maturation or immunoglobulinproduction in an animal using BAFF-R polypeptide. Also included aremethods of stimulating B-cell growth, dendritic cell-induced B-cellgrowth and maturation or immunoglobulin production in an animal usingBAFF-R polypeptide or co-stimulating B-cell growth, dendriticcell-induced B-cell growth and maturation or immunoglobulin productionin an animal using BAFF-R polypeptide and an anti-T antibody, a CD40ligand or an anti-CD40 ligand.

In another embodiment, the invention provides methods of using BAFF-R inthe treatment of autoimmune diseases, hypertension, cardiovasculardisorders, renal disorders, B-cell lympho-proliferate disorders,immunosuppressive diseases, organ transplantation, and HIV. Alsoincluded are methods of using agents for treating, suppressing oraltering an immune response involving a signaling pathway between BAFF-Rand its ligand, and methods of inhibiting inflammation by administeringan antibody specific for a BAFF-R or an epitope thereof.

The methods of the present invention are preferably carried out byadministering a therapeutically effective amount of a BAFF-Rpolypeptide, a chimeric molecule comprising a BAFF-R polypeptide fusedto a heterologous amino acid sequence, or an anti-BAFF-R antibodyhomolog.

In one embodiment, the invention provides pharmaceutical compositionscomprising a BAFF-R polypeptide and a pharmaceutically acceptableexcipient.

In another embodiment, the invention provides chimeric moleculescomprising BAFF-R polypeptide fused to a heterologous polypeptide oramino acid sequence. An example of such a chimeric molecule comprises aBAFF-R fused to a Fc region of an immunoglobulin or an epitope tagsequence.

In another embodiment, the invention provides an antibody thatspecifically binds to a BAFF-R polypeptide. Optionally, the antibody isa monoclonal antibody

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleic acid sequence (SEQ ID NO:2) of a cDNA for humanBAFF-R (BCMA) and its derived amino acid sequence (SEQ ID NO: 1).Potential start of translation at either nucleic acid residue 219 or228; cysteine rich domain (CRD) at nucleic acid residues 240-341 of SEQID NO:2 (amino acid residues 8-41 of SEQ ID NO:1); and potentialtransmembrane region at nucleic acid residues 375-459 of SEQ ID NO:2.

FIGS. 2A and 2B show the nucleic acid sequence (SEQ ID NO:4) and itsderived amino acid sequence (SEQ ID NO:3) of pJST538, a plasmid encodingBAFF-R-Fc: nucleic acid residues 1-69, murine IgG-kappa signal sequence;nucleic acid residues 70-222, BAFF-R (nucleic acid residues1-153);nucleic acid residues 223-906, human IgG.

FIG. 3 shows the nucleic acid sequence of pJST535, a plasmid encoding afull length human BAFF-R and its derived amino acid sequence.

FIG. 4 shows a structure comparison between TNF-R55 and BAFF-R.

FIGS. 5A and 5B show 293EBNA cells transfected with either (FIG. 5A)CH269 (1.0 ug) or (FIG. 5B) pJST535 (0.1 ug), the plasmid expressingfull length BAFF-R, and stained with 0.5 ug/ml flag-hBAFF in the plateassay format.

FIG. 6A shows FACS overlay of 293EBNA transfected with pJST535 andstained as follows: no ligand (black histogram), 1 ug/ml flag-hCD40L(pink) or flag-hBAFF (green). All samples were then stained withanti-flag M2 followed by donkey anti-mouse IgG as described in methodsin Example 2.

FIGS. 6B-6J show FACS histograms with statistics of same experiment.Staining is as follows: (FIG. 6B) unstained, (FIG. 6C) 7AAD only, (FIG.6D) 2^(nd) step and 7AAD only, (FIG. 6E) 9 ug/ml flag-hBAFF, (FIG. 6F) 3ug/ml flag-hBAFF, (FIG. 6G) 1 ug/ml flag-hBAFF, (FIG. 6H) 0.33 ug/mlflag-hBAFF, (FIG. 6I) 0.11 ug/mi flag-hBAFF, (FIG. 6J) flag-hCD40L 1ug/ml.

FIG. 7 shows immunoprecipitations with BAFF-R-Fc as described in methodsin Example 4, Molecular weight standards in kDa are as labeled to theleft of the figure. Lane (1) 12.5 ng flag-hTWEAK, (2) 12.5 ngflag-hBAFF, (3) immunoprecipitation of flag-hBAFF by 0.5 ml BAFF-R-Fcconditioned media, (4) immunoprecipitation of flag-hTWEAK by 0.5 mlBAFF-R-Fc conditioned media, (5) immunoprecipitation of no ligand by 0.5ml BAFF-R-Fc conditioned media, (6) immunoprecipitation of flag-hBAFF by5 ml conditioned media from untransfected 293EBNA, (7)immunoprecipitation of flag-hTWEAK by 0.5 ml conditioned media fromuntransfected 293EBNA.

FIG. 8 shows a plot of the results of a splenocyte proliferation assayin which the counts per minute (CPM) incorporated into mouse splenocytecells is plotted against the amount of human BAFF added (ug/ml).

FIG. 9 shows a plot of the results of a BAFF blocking assay analyzingBAFF binding to Raji cells in which the MFI (mean fluorescenceintensity) readings are plotted against the amount of R:hIgG1 (ng/ml).

FIG. 10A shows plots of expression of IgM vs. CD1 in a FACS analysis forBaff Tg mice that received h-Ig (middle panel) or hBCMA-Ig (lower panel)and for wildtype littermate controls that received PBS injections (upperpanel), as described in Example 11.

FIG. 10B shows plots of the expression of CD21 vs. IgM in FACS analysisby gating on IgD positive populations for Baff Tg mice that receivedh-Ig (middle panel) or hBCMA-Ig (lower panel) and for wildtypelittermate controls that received PBS injections (upper panel), asdescribed in Example 11.

FIG. 10C shows plots of the expression of CD21 vs. IgM in FACS analysisby gating on IgD negative populations for Baff Tg mice that receivedh-Ig (middle panel) or hBCMA-Ig (lower panel) and for wildtypelittermate controls that received PBS injections (upper panel), asdescribed in Example 11.

FIG. 11 shows spleen weights for all groups of Baff Tg mice (given asweight in mg+/−standard deviation).

FIG. 12 shows a plot of proteinurea (mg/dL) vs. injection number forBaff Tg mice treated with PBS, hIg, hBCMA-Ig and for littermatecontrols.

FIG. 13 shows a plot of the average mean arterial pressure (mmHg) inBaff Tg mice and wildtype controls.

FIG. 14 shows a plot of the individual mean arterial pressure (mmHg) forBaff Tg mice and wildtype controls.

FIG. 15 shows a bar graph of the percentage of SNF1 mice that displaysevere nephritis after treatment with BAFF-R-Ig (BCMA-Ig), HuIgG or PBS.

FIG. 16 shows a graph of the total CD11c+DC cell number (in millions)for mice treated in vivo with 20 ug BCMA-Ig, 50 ug BCMA-Ig, HuIgG orPBS. CD11c+ DC cell populations examined were: (1) CD8a− CD4−, (2)CD8a+CD4−, and (3) CD8a− CD4+.

DETAILED DESCRIPTION I. Definitions

The terms “BAFF-R” and “BCMA” when used herein encompass native sequenceBAFF-R and BAFF-R variants (which are further defined herein). TheBAFF-R may be isolated from a variety of sources, such as from murine orhuman tissue types or from another source, or prepared by recombinant orsynthetic methods.

A “native sequence BAFF-R” comprises a polypeptide having the same aminoacid sequence as BAFF-R derived from nature. Such native sequence BAFF-Rcan be isolated from nature or can be produce by recombinant orsynthetic means. The naturally-occurring truncated or secreted forms ofthe BAFF-R (e.g. soluble forms containing for instance, an extracellulardomain sequence), naturally-occurring variant forms (e.g., alternativelyspliced forms) and naturally-occurring allelic variants of the BAFF-R.In one embodiment of the invention, the native sequence BAFF-R is amature or full-length native sequence BAFF-R polypeptide comprisingamino acids 1 to 184 of SEQ ID NO: 1 or fragment thereof.

The “BAFF-R extracellular domain” or “BAFF-R ECD” refers to a form ofBAFF-R which is essentially free of transmembrane and cytoplasmicdomains of BAFF-R. Ordinarily, BAFF-R extracellular domain will haveless than 1% of such transmembrane and cytoplasmic domains and willpreferably have less than 0.5% of such domains. Optionally, BAFF-R ECDwill comprise amino acid residues 8 to 41 of SEQ ID NO:1, or amino acidresidues 4 to 51 of SEQ ID NO: 1, or amino acid residues 1 to 53 of SEQID NO: 1. In a preferred embodiment of the present invention, the BAFF-RECD comprises amino acid residues 1 to 51 of SEQ ID NO: 1. It will beunderstood by the skilled artisan that the transmembrane domainidentified for the BAFF-R polypeptide of the present invention isidentified pursuant to criteria routinely employed in the art foridentifying that type of hydrophobic domain. The exact boundaries of atransmembrane domain may vary but most likely by no more than about 5amino acids at either end of the domain specifically mentioned herein.Accordingly, the BAFF-R ECD may optionally comprise amino acids 8-41(SEQ ID NO:1).

“BAFF-R variant” means an active BAFF-R as defined below having at leastabout 80% amino acid sequence identity with the BAFF-R having thededuced amino acid sequence shown in SEQ ID NO: 1 for a full-lengthnative sequence BAFF-R or with a BAFF-R ECD sequence. Such BAFF-Rvariants include, for instance, BAFF-R polypeptides wherein one or moreamino acid residues are added, or deleted, at the end or C-terminus ofthe sequence of SEQ ID NO: 1. Ordinarily, a BAFF-R variant will have atleast about 80% or 85% amino acid sequence identity, more preferably atleast about 90% amino acid sequence identity, and even more preferablyat least about 95% amino acid sequence identity with the amino acidsequence of SEQ ID NO:1.

“Percent (%) amino acid sequence identity” with respect BAFF-R sequencesidentified herein is defined as the percentage of amino acid residues ina candidate sequence that are identical with the amino acid residues inthe BAFF-R sequence, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publically available computersoftware such as BLAST, ALIGN, or Megalign (DNASTAR) software. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximum alignmentover the full length of the sequences being compared.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising BAFF-R, or a domain sequence thereof, fused to a“tag polypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, or which can beidentified by some other agent, yet is short enough so that it does notinterfere with activity of the BAFF-R. The tag polypeptide preferablyalso is fairly unique so that the antibody does not substantiallycross-react with other epitopes. Suitable tag polypeptides generallyhave at least 6 amino acid residues and usually between about 8 to about50 amino acid residues (preferably, about 10 to about 20 residues).

“Isolated” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminatecomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity SDSPAGE undernon-reducing or reducing conditions using Coomassie blue or preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the BAFF-R's naturalenvironment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

The term “antibody” is used in the broadest sense and specificallycovers single BAFF-R monoclonal antibodies (including agonist,antagonist, and neutralizing antibodies) and anti-BAFF-R antibodycompositions with polyepitopic specificity. The term “monoclonalantibody” as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e. the individualantibodies comprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.

A “purified preparation” or a “substantially pure preparation” of apolypeptide, as used herein, means a polypeptide that has been separatedfrom other proteins, lipids, and nucleic acids with which it naturallyoccurs. Preferably, the polypeptide is also separated from othersubstances, e.g., antibodies, matrices, etc., which are used to purifyit.

The terms, “treating”, “treatment” and “therapy” as used herein refersto curative therapy, prophylactic therapy, and preventative therapy.

The terms “peptides”, “proteins”, and “polypeptides” are usedinterchangeably herein.

“Biologically active” as used herein, means having an in vivo or invitro activity which may be performed directly or indirectly.Biologically active fragments of BAFF-R may have, for example, 70% aminoacid homology with the active site of the receptor, more preferably atleast 80%, and most preferably, at least 90% homology. Identity orhomology with respect to the receptor is defined herein as thepercentage of amino acid residues in the candidate sequence which areidentical to the BAFF-R residues in SEQ ID NO: 1, or which are identicalto a defined portion of the amino acid residues in SEQ ID NO:1.

The term “mammal” as used herein refers to any animal classified as amammal including humans, cows, horses, dogs, mice and cats. In preferredembodiment of the invention, the mammal is a human.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare described in the literature.

Reference will now be made in detail to the present preferredembodiments of the invention. This invention relates to the use ofBAFF-R and BAFF-R related molecules to effect the growth and maturationof B-cells and the secretion of immunoglobulin. The invention relates tothe use of BAFF-R and BAFF-R related molecules to effect responses ofthe immune system, as necessitated by immune-related disorders.Additionally, this invention encompasses the treatment of cancer andimmune disorders through the use of a BAFF-R, or BAFF-R related genethrough gene therapy methods.

The BAFF-R and homologs thereof produced by hosts transformed with thesequences of the invention, as well as native BAFF-R purified by theprocesses known in the art, or produced from known amino acid sequences,are useful in a variety of methods for anticancer, antitumor andimmunoregulatory applications. They are also useful in therapy andmethods directed to other diseases.

Another aspect of the invention relates to the use of the polypeptideencoded by the isolated nucleic acid encoding the BAFF-R in “antisense”therapy. As used herein, “antisense” therapy refers to administration orin situ generation of oligonucleotides or their derivatives whichspecifically hybridize under cellular conditions with the cellular mRNAand/or DNA encoding the ligand of interest, so as to inhibit expressionof the encoded protein, i.e. by inhibiting transcription and/ortranslation. The binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” therapy refers to a range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid, which, when transcribed in the cell,produces RNA which is complementary to at least a portion of thecellular mRNA which encodes BAFF-ligand. Alternatively, the antisenseconstruct can be an oligonucleotide probe which is generated ex vivo.Such oligonucleotide probes are preferably modified oligonucleotideswhich are resistant to endogenous nucleases, and are therefor stable invivo. Exemplary nucleic acids molecules for use as antisenseoligonucleotides are phosphoramidates, phosphothioate andmethylphosphonate analogs of DNA (See, e.g., U.S. Pat. Nos. 5,176,996;5,264,564; and 5,256,775). Additionally, general approaches toconstructing oligomers useful in antisense therapy have been reviewed,for example, by Van Der Krol et al., (1988) Biotechniques 6:958-976; andStein et al. (1988) Cancer Res 48: 2659-2668, specifically incorporatedherein by reference.

The BAFF-R of the invention, as discussed above, is a member of the TNFreceptor family. The protein, fragments or homologs thereof may havewide therapeutic and diagnostic applications.

The polypeptides of the invention specifically interact with BAFF, apolypeptide previously described in WO99/12964 incorporated by referenceherein. However, the peptides and methods disclosed herein enable theidentification of molecules which specifically interact with the BAFF-Ror fragments thereof.

The claimed invention in certain embodiments includes methods of usingpeptides derived from BAFF-R which have the ability to bind to BAFF.Fragments of the BAFF-Rs can be produced in several ways, e.g.,recombinantly, by PCR, proteolytic digestion or by chemical synthesis.Internal or terminal fragments of a polypeptide can be generated byremoving one or more nucleotides from one end or both ends of a nucleicacid which encodes the polypeptide. Expression of the mutagenized DNAproduces polypeptide fragments.

Chimeric molecules for use in the present invention can also be producedusing techniques known in the art. The present invention contemplatesthe use of chimeric molecules comprising a BAFF-R polypeptide (orvariant thereof) fused to a heterologous amino acid sequence, such asthe IgG Fc domain of an immunoglobulin. Preferably, such chimericmolecules are soluble and comprise a soluble BAFF-R polypeptide.

Polypeptide fragments can also be chemically synthesized usingtechniques known in the art such as conventional Merrifield solid phasef-moc or t-boc chemistry. For example, peptides and DNA sequences of thepresent invention may be arbitrarily divided into fragments of desiredlength with no overlap of the fragment, or divided into overlappingfragments of a desired length. Methods such as these are described inmore detail below.

Generation of Soluble Forms of BAFF-R

Soluble forms of the BAFF-R can often signal effectively and hence canbe administered as a drug which now mimics the natural membrane form. Itis possible that the BAFF-R claimed herein are naturally secreted assoluble cytokines, however, if not, one can reengineer the gene to forcesecretion. To create a soluble secreted form of BAFF-R, one would removeat the DNA level the N-terminus transmembrane regions, and some portionof the stalk region, and replace them with a type I leader oralternatively a type II leader sequence that will allow efficientproteolytic cleavage in the chosen expression system. A skilled artisancould vary the amount of the stalk region retained in the secretionexpression construct to optimize both ligand binding properties andsecretion efficiency. For example, the constructs containing allpossible stalk lengths, i.e. N-terminal truncations, could be preparedsuch that proteins starting at amino acids 1 to 51 would result. Theoptimal length stalk sequence would result from this type of analysis.

In preferred embodiments of the present invention, the soluble BAFF-Rpolypeptide is: an isolated native sequence BAFF-R polypeptidecomprising amino acid residues 1 to 51 of SEQ ID NO:1 or a fragmentthereof; an isolated BAFF-R polypeptide having at least 80% (and morepreferably 90%) amino acid sequence identity with native sequence BAFF-Rpolypeptide comprising amino acid residues 1 to 51 of SEQ ID NO: 1 or afragment thereof; or an isolated BAFF-R polypeptide comprising aminoacid residues 8 to 41 of SEQ ID NO: 1 or a fragment thereof.

Generation of Antibodies Reactive with the BAFF-R

The invention also includes antibodies specifically reactive with theclaimed BAFF-R or its co-receptors. Anti-protein/anti-peptide antiseraor monoclonal antibodies can be made by standard protocols (See, forexample, Antibodies: A Laboratory Manual ed. by Harlow and Lane (ColdSpring Harbor Press: 1988)). A mammal such as a mouse, a hamster orrabbit can be immunized with an immunogenic form of the peptide.Techniques for conferring immunogenicity on a protein or peptide includeconjugation to carriers, or other techniques, well known in the art.

An immunogenic portion of BAFF-R or its co-receptors can be administeredin the presence of an adjuvant. The progress of immunization can bemonitored by detection of antibody titers in plasma or serum. StandardELISA or other immunoassays can be used with the immunogen as antigen toassess the levels of antibodies.

In a preferred embodiment, the subject antibodies are immunospecific forantigenic determinants of BAFF-R or its co-receptors, e.g. antigenicdeterminants of a polypeptide of SEQ ID NO:1, or a closely related humanor non-human mammalian homolog (e.g. 70, 80 or 90 percent homologous,more preferably at least 95 percent homologous). In yet a furtherpreferred embodiment of the present invention, the anti-BAFF-R oranti-BAFF-co-receptor antibodies do not substantially cross react (i.e.react specifically) with a protein which is e.g., less than 80 percenthomologous to SEQ ID NO:1; preferably less than 90 percent homologouswith SEQ ID NO: 1; and, most preferably less than 95 percent homologouswith SEQ ID NO: 1. By “not substantially cross react”, it is meant thatthe antibody has a binding affinity for a non-homologous protein whichis less than 10 percent, more preferably less than 5 percent, and evenmore preferably less than 1 percent, of the binding affinity for aprotein of SEQ ID NO: 1.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with BAFF-R, or itsreceptors. Antibodies can be fragmented using conventional techniquesand the fragments screened for utility in the same manner as describedabove for whole antibodies. For example, F(ab′)₂ fragments can begenerated by treating antibody with pepsin. The resulting F(ab′)₂fragment can be treated to reduce disulfide bridges to produce Fab′fragments. The antibodies of the present invention are further intendedto include biospecific and chimeric molecules having anti-BAFF-R oranti-BAFF-co-receptor activity. Thus, both monoclonal and polyclonalantibodies (Ab) directed against BAFF-R, and their co-receptors, andantibody fragments such as Fab′ and F(ab′)₂, can be used to block theaction of the BAFF-R and its respective co-receptors.

Various forms of antibodies can also be made using standard recombinantDNA techniques. (Winter and Milstein, Nature 349: 293-299 (1991)specifically incorporated by reference herein.) For example, chimericantibodies can be constructed in which the antigen binding domain froman animal antibody is linked to a human constant domain (e.g. Cabilly etal., U.S. Pat. No. 4,816,567, incorporated herein by reference).Chimeric antibodies may reduce the observed immunogenic responseselicited by animal antibodies when used in human clinical treatments.

In addition, recombinant “humanized antibodies” which recognize BAFF-Ror its co-receptors can be synthesized. Humanized antibodies arechimeras comprising mostly human IgG sequences into which the regionsresponsible for specific antigen-binding have been inserted. Animals areimmunized with the desired antigen, the corresponding antibodies areisolated, and the portion of the variable region sequences responsiblefor specific antigen binding are removed. The animal-derived antigenbinding regions are then cloned into the appropriate position of humanantibody genes in which the antigen binding regions have been deleted.Humanized antibodies minimize the use of heterologous (i.e. interspecies) sequences in human antibodies, and thus are less likely toelicit immune responses in the treated subject.

Construction of different classes of recombinant antibodies can also beaccomplished by making chimeric or humanized antibodies comprisingvariable domains and human constant domains (CH1, CH2, CH3) isolatedfrom different classes of immunoglobulins. For example, antibodies withincreased antigen binding site valencies can be recombinantly producedby cloning the antigen binding site into vectors carrying the human:chain constant regions. (Arulanandam et al., J. Exp. Med., 177:1439-1450 (1993), incorporated herein by reference.)

In addition, standard recombinant DNA techniques can be used to alterthe binding affinities of recombinant antibodies with their antigens byaltering amino acid residues in the vicinity of the antigen bindingsites. The antigen binding affinity of a humanized antibody can beincreased by mutagenesis based on molecular modeling. (Queen et al.,Proc. Natl. Acad. Sci. 86: 10029-33 (1989) incorporated herein byreference.

Generation of Analogs: Production of Altered DNA and Peptide Sequences

Analogs of the BAFF-R can differ from the naturally occurring BAFF-R inamino acid sequence, or in ways that do not involve sequence, or both.Non-sequence modifications include in vivo or in vitro chemicalderivatization of the BAFF-R. Non-sequence modifications include, butare not limited to, changes in acetylation, methylation,phosphorylation, carboxylation or glycosylation.

Preferred analogs include BAFF-R biologically active fragments thereof,whose sequences differ from the sequence given in SEQ ID NO: 1, by oneor more conservative amino acid substitutions, or by one or morenon-conservative amino acid substitutions, deletions or insertions whichdo not abolish the activity of BAFF-ligand. Conservative substitutionstypically include the substitution of one amino acid for another withsimilar characteristics, e.g. substitutions within the following groups:valine, glycine; glycine, alanine; valine, isoleucine, leucine; asparticacid, glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and, phenylalanine, tyrosine.

Uses

The full length BAFF-R gene (SEQ ID NO: 2) or portions thereof may beused as hybridization probes for a cDNA library to isolate, forinstance, still other genes which have a desired sequence identity tothe BAFF-R sequence disclosed in SEQ ID NO: 2. Nucleotide sequencesencoding BAFF-R can also be used to construct hybridization probes formapping the gene which encodes the BAFF-R and for the genetic analysisof individuals with genetic disorders. Screening assays can be designedto find lead compounds that mimic the biological activity of a BAFF-R.Such screening assays will include assays amenable to high-throughputscreening of chemical libraries, making them particularly suitable foridentifying small molecule drug candidates. Small molecules contemplatedinclude synthetic organic or inorganic compounds. Nucleic acids whichencode BAFF-R or its modified forms can also be used to generate eithertransgenic animals or “knock out” animals which in turn are useful inthe development and screening of therapeutically useful reagents.

As described herein, in one embodiment of the invention, there areprovided methods of stimulating B-cell growth, dendritic cell-inducedB-cell growth and maturation or immunoglobulin production in an animalusing BAFF-R polypeptide or co-stimulating B-cell growth, dendriticcell-induced B-cell growth and maturation or immunoglobulin productionin an animal using BAFF-R polypeptide and an anti-T antibody, a CD40ligand or an anti-CD40 ligand. Also included are methods of inhibitingB-cell growth, dendritic cell-induced B-cell growth and maturation orimmunoglobulin production in an animal using BAFF-R polypeptide.

In another embodiment, the invention provides methods of using BAFF-R inthe treatment of autoimmune diseases, hypertension, cardiovasculardisorders, renal disorders, B-cell lympho-proliferate disorders,immunosuppressive diseases, organ transplantation, inflammation, andHIV. Also included are methods of using agents for treating, suppressingor altering an immune response involving a signaling pathway betweenBAFF-R and its ligand.

In one embodiment, the invention provides pharmaceutical compositionscomprising a BAFF-R polypeptide and a pharmaceutically acceptableexcipient. Suitable carriers for a BAFF-R polypeptide, for instance, andtheir formulations, are described in Remington' Pharmaceutical Sciences,16^(th) ed., 1980, Mack Publishing Co., edited by Oslo et al. Typicallyan appropriate amount of a pharmaceutically acceptable salt is used inthe formulation to render the formulation isotonic. Examples of thecarrier include buffers such as saline, Ringer's solution and dextrosesolution. The pH of the solution is preferably from about 5 to about 8,and more preferably from about 7.4 to about 7.8. Further carriersinclude sustained release preparations such as semipermeable matrices ofsolid hydrophobic polymers, which matrices are in the form of shapedarticles, e.g. liposomes, films or microparticles. It will be apparentto those of skill in the art that certain carriers may be morepreferable depending upon for instance the route of administration andconcentration of the a BAFF-R polypeptide being administered.

Administration may be accomplished by injection (eg intravenous,intraperitoneal, subcutaneous, intramuscular) or by other methods suchas infusion that ensure delivery to the bloodstream in an effectiveform.

Practice of the present invention will employ, unless indicatedotherwise, conventional techniques of cell biology, cell culture,molecular biology, microbiology, recombinant DNA, protein chemistry, andimmunology, which are within the skill of the art. Such techniques aredescribed in the literature. See, for example, Molecular Cloning: ALaboratory Manual, 2nd edition. (Sambrook, Fritsch and Maniatis, eds.),Cold Spring Harbor Laboratory Press, 1989; DNA Cloning, Volumes I and II(D. N. Glover, ed), 1985; Oligonucleotide Synthesis, (M. J. Gait, ed.),1984; U.S. Pat. No. 4,683,195 (Mullis et al.); Nucleic AcidHybridization (B. D. Hames and S. J. Higgins, eds.), 1984; Transcriptionand Translation (B. D. Hames and S. J. Higgins, eds.), 1984; Culture ofAnimal Cells (R. I. Freshney, ed). Alan R. Liss, Inc., 1987; ImmobilizedCells and Enzymes, IRL Press, 1986; A Practical Guide to MolecularCloning (B. Perbal), 1984; Methods in Enzymology, Volumes 154 and 155(Wu et al., eds), Academic Press, New York; Gene Transfer Vectors forMammalian Cells (J. H. Miller and M P. Calos, eds.), 1987, Cold SpringHarbor Laboratory; Immunochemical Methods in Cell and Molecular Biology(Mayer and Walker, eds.), Academic Press, London, 1987; Handbook ofExperiment Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds.), 1986; Manipulating the Mouse Embryo, Cold Spring HarborLaboratory Press, 1986.

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof.

EXAMPLES Example 1: Detection of BAFF Binding to BAFF-R Using a PlateAssay

In this example, the binding of BAFF to BAFF-R transfected cells using aplate assay is described.

Full-length human BAFF-R was generated from BJAB polyA+RNA using theSuperscriptII preamplification kit (Life Technologies) to generate thecDNA template and amplified by Pfu1 using primers complementary to the5′ and 3′ coding sequences of BAFF-R. The PCR product was cloned intoCH269, a derivative of pCEP4 (Invitrogen). The resultant clone wastermed pJST535. Human embryonic kidney cells containing the EBNA-1 gene(293EBNA) were seeded into 6 well plates coated with fibronectin andtransfected by lipofectamine (Life Technologies) with either pJST535 atvarious dilutions or CH269 as a background control. At 48 hrs posttransfection, transfected cells were assayed for their ability to bindsoluble flag-hBAFF (amino acids L83-L285) as follows. All incubationswere at room temperature. Conditioned media was aspirated from the wellsand the cells washed with BHA buffer (20 mM HEPES pH7.0, 0.5 mg/ml BSA,0.1% NaN3) and incubated with 0.5 ug/ml FLAG-hBAFF diluted in PBScontaining 1 mM MgCl2, 1 mM CaCl2 and 0.1% NaN3. After an 1 hr.incubation, the BAFF solution was removed and the cells were washed withBHA. The cells were next incubated for 30 min. in a PBS solutioncontaining 1 ug/ml of the anti-FLAG monoclonal antibody, M2 (Sigma).This solution was aspirated and the cells were washed with BHA. Thecells were then incubated for 30 min, in a 1:3000 dilution of thealkaline phosphatase conjugated goat anti-mouse IgG F(ab)′2 (JacksonImmunoResearch). This solution was aspirated and the cells washed withBHA. To reduce the amount of background staining due to endogenousalkaline phosphatase, the cells were incubated for 15 min. in 2.5 mMlevamisol (Vector Laboratories) diluted in 100 mM NaCl, 100 mM Tris-ClpH9.5 and 5 mM MgCl2. For chromogenic detection of alkaline phosphatase,the inhibitor solution was aspirated and the cells were incubated with asolution of fast red and napthol phosphate (Pierce). Staining wasobserved and photographed through a low power microscope.

The deposition of fast red dye was observed for all the wellstransfected with the BAFF-R expressing plasmid, pJST535. The frequencyof the signal titrated away as the amount of plasmid transfected intothe cells decreased. No staining was observed for the control expressionvector, CH269, transfected cells. Also, no staining was observed on anyof the transfected cells when FLAG-hBAFF was omitted from the stainingprotocol or when another TNF family member ligand, FLAG-tagged LIGHT,was substituted for flag-hBAFF. Therefore the staining of BAFF-Rtransfected cells with BAFF appears specific.

Example 2: BAFF Binding to BAFF-R Transfected Cells by FACS Analysis

This example describes the detection of BAFF to BAFF-R transfected cellsusing FACS analysis.

The plasmid encoding full-length BAFF-R, pJST535, was transfected into293EBNA cells using FuGene6 (Boehringer Mannheim). At 24 or 48 hr posttransfection, cells were removed from the plates using 5 mM EDTA in PBSand counted. The cells were washed twice with FACS buffer (PBScontaining 10% fetal bovine serum, 0.1% NaN3 and 10 ug/ml hIgG (JacksonImmunoResearch) and then 2.5×10⁵ cells were incubated for 1 hr on icewith FLAG-hBAFF diluted into FACS buffer at concentrations ranging from9 ug/ml to 0.037 ug/ml. The cells were washed with FACS buffer andincubated with the anti-FLAG monoclonal antibody, M2, at 5 ug/ml for 30min. on ice. The cells were washed with FACS buffer and incubated for 30min. on ice in a solution containing a 1:100 dilution of R-phycoerythrinconjugated F(ab′)2 donkey anti-mouse IgG and 10 ug/ml 7-AAD. After thecells were washed with FACS buffer, they were resuspended in FACS buffercontaining 1% paraformaldehyde. FACS analysis followed where the 7-AADpositive (dead) cells were gated out.

The results of the FACS analysis indicate that a fraction of the cellsthat were transfected with BAFF-R are able to bind BAFF. At a BAFFconcentration of 9 ug/ml, about 28% of the cells bind BAFF for a meanfluorescence intensity (MFI) of 366. Using the PE-labeled donkeyanti-mouse reagent alone, there is not a significant shift of the cells.Only 1.3% of the cells have a MFI of 23.5 with the average of all thecells being 5.5. The signal on the BAFF-R transfected cells titrates outwith decreasing amounts of BAFF. At 100 ng/ml, 8.76% of the cells havean MFI of 78.9.

Example 3: FACS Analysis of BAFF/BAFF-R Interaction Including a GFPMarker

In this example, the ability of BAFF to bind to cells co-transfectedwith BAFF-R and a GFP reporter plasmid is described.

The 293EBNA cells were transfected with pJST535 and a GFP reporterplasmid, as described in example 2. The reporter plasmid encodes amembrane anchored GFP molecule. Using co-transfection of the twoplasmids, we analyzed the percentage of transfected cells which werecapable of binding BAFF. The cells were removed from the plates andsubjected to BAFF binding and detection as described in example 2.Again, 7-AAD was included in order to gate out the dead cells. Thesamples were analyzed by FACS and plotted. The upper right quadrantrepresents cells with bound BAFF (phycoerythrin positive) and expressingGFP.

Although not all of the GFP transfected cells appear to bind BAFF, thereis a significant fraction of the cells in the upper right quadrantcompared to the control. Thirty-three percent of the transfected cellsare in the upper right compared to 8 percent It may be that a certainlevel of BAFF-R expression is required for BAFF to bind to the cells. Itis also possible that a co-receptor is required for a high affinityinteraction and that this receptor is limiting on the 293EBNA cells.

Example 4: Immunoprecipitation of Flag-hBAFF by BAFF-R-Fc Fusion

This example describes specific interaction of flag-hBAFF withBAFF-R-Fc, a molecule composed of the Cysteine Rich Domain (CRD) ofBAFF-R fused to the Fc domain of human IgG1.

The CRD of BAFF-R was generated by RT-PCR from BJAB polyA+RNA as inExample 1 using as a 3′ primer an oligo complementary to nucleotides132-152 of the hBAFF-R coding sequence. The resultant PCR fragment wascloned in CH269 downstream of a murine IgG-kappa signal sequence andupstream of the Fc moiety of human IgG. This construct was termedpJST538. The construct pJST538 or CH269 were transfected to 293EBNA bylipofectamine. Conditioned media and cell extracts were harvested at 20hours post transfection. Cells were solublized in 20 mM Tris pH7.5/50 mMNacl/0.5% NP40/0.5% deoxycholic acid and debris spun out. An aliquot ofthe conditioned media and cell extracts were combined with an equalvolume 2×SDS reducing buffer, boiled and subjected to SDS-PAGE andWestern transfer. To verify expression, the membrane was probed with1:3000 dilution of mouse anti-human IgG conjugated to horse radishperoxidase (HRP) in 5% nonfat dry milk in TBST at room temperature for30 minutes and washed with TBST. Blots were developed with ECL(Amersham) and exposed to film.

Immunoprecipitations were performed by incubating 25 ng of eitherflag-hBAFF or flag-hTWEAK with 0.5 ml of conditioned media from 293EBNAtransfected with either pJST538 or CH269 at 4° C. for 1 hour withagitation followed by the addition of 30 ul ProteinA-Sepharose(Pharmacia) and continued agitation overnight. ProteinA-Sepharose beadswere washed twice with PBS and resuspended in 2×SDS reducing samplebuffer. After SDS-PAGE and western transfer, the blots were incubatedwith 5 ug/ml anti-flag monoclonal antibody M2 (Sigma) in 5% nonfat drymilk in TBST at room temperature for 1 hour. Blots washed with TBST andincubated with a 1:3000 dilution of goat anti-mouse IgG HRP conjugate(Jackson Immunoresearch) in 5% nonfat dry milk in TBST at roomtemperature for 30 minutes. Blots were developed with ECL (Amersham) andexposed to film.

Upon transfection of pJST538 to 293EBNA cells, expression of anapproximately 43 kDa protein was detected in both the cell extract andconditioned medium by Western blot analysis with mouse anti-human IgG(Jackson Immunoresearch), indicating that the BAFF-R-Fc fusion wasefficiently expressed and secreted.

In the immunoprecipitations a band was observed only as a result of theincubation of BAFF-R-Fc and flag-hBAFF. This band co-migrated with adirectly loaded sample of flag-hBAFF. None of the other lanes produced asignal, indicating that the interaction between BAFF-R-Fc and flag-hBAFFis specific.

Example 5: Generation of Soluble Receptor Forms

To form an receptor inhibitor for use in man, one requires the humanreceptor cDNA sequence of the extracellular domain. If the mouse form isknown, human cDNA libraries are screened using the mouse cDNA sequenceand such manipulations are routinely carried out in this area. With ahuman cDNA sequence, one can design oligonucleotide primers to PCRamplify the extracellular domain of the receptor in the absence of thetransmembrane and intracellular domains. Typically, one includes most ofthe amino acids between the last disulfide linked “TNF domain” and thetransmembrane domain. One can vary the amount of “stalk” region includedto optimize the potency of the resultant soluble receptor. Thisamplified piece would be engineered to include suitable restrictionsites to allow cloning into various C-terminal Ig fusion chimeravectors. Alternatively, one can insert a stop signal at the 3′ end andmake a soluble form of the receptor without resorting to the use of a Igfusion chimera approach. The resultant vectors can be expressed in mostsystems used in biotechnology including yeast, insect cells, bacteriaand mammalian cells and examples exist for all types of expression.Various human Fc domains can be attached to optimize or eliminate FcRand complement interactions as desired. Alternatively, mutated forms ofthese Fc domains can be used to selectively remove FcR or complementinteractions or the attachment of N-linked sugars to the Fc domain whichhas certain advantages.

Example 6: Generation of Agonistic or Antagonistic Antibodies

The above described soluble receptor forms can be used to immunize miceand to make monoclonal antibodies by conventional methods. The resultantmAbs that are identified by ELISA methods can be further screened foragonist activity either as soluble antibodies or immobilized on plasticin various in vitro cellular assays. Often the death of the HT29 cellline is a convenient system that is sensitive to signaling through manyTNF receptors. If this line does not possess the receptor of interest,that full length receptor can be stably transfected into the HT29 lineto now allow the cytotoxicity assay to work.

Alternatively, such cells can be used in the Cytosensor apparatus toassess whether activation of the receptor can elicit a pH change that isindicative of a signaling event. TNF family receptors signal well insuch a format and this method does not require one to know the actualbiological events triggered by the receptor. The agonistic mAbs would be“humanized” for clinical use. This procedure can also be used to defineantagonistic mAbs. Such mAbs would be defined by the lack of agonistactivity and the ability to inhibit receptor-ligand interactions asmonitored by ELISA, classical binding or BIAcore techniques. Lastly, theinduction of chemokine secretion by various cells in response to anagonist antibody can form a screening assay.

Example 7: Screening for Inhibitors of the Receptor-Ligand Interaction

Using the receptor-Ig fusion protein, one can screen eithercombinatorial libraries for molecules that can bind the receptordirectly. These molecules can then be tested in an ELISA formatted assayusing the receptor-Ig fusion protein and a soluble form of the ligandfor the ability to inhibit the receptor-ligand interaction. This ELISAcan be used directly to screen various natural product libraries etc.for inhibitory compounds. The receptor can be transfected into a cellline such as the HT29 line to form a biological assay (in this casecytotoxicity) that can then form the screening assay.

Example 8: BAFF-R-IgG Causes a Reduction in the Number of B Cells inNormal Mice

Eight-week-old female BALB/c mice were purchased from The JacksonLaboratory (Bar Harbor, Me.). Mice (3/group) received i.p. either PBS,400 μg of human BAFF-R-huIgG1 (hBAFF-R-Ig) fusion protein (supplied byTeresa Cachero, Biogen), or 400 μg of purified human IgG (HuIgG)(Sandoz, Basel, Switzerland) on days −8, −5, −1 and +2. Mice received100 μl of 10% sheep red blood cells (SRBC) (Colorado Serum Company,Denver, Colo.) on day 0.

At the time of sacrifice, blood was collected via cardiac puncture intotubes containing EDT, and red blood cells were lysed in a hypotonicbuffer. Blood was also collected without EDTA for serum preparation.Single cell suspensions were prepared from spleens and mesenteric lymphnodes (MLN) and red blood cells were lysed in a hypotonic buffer. Flowcytometry was performed using PE-conjugated anti-CD45R/B220,anti-syndecan/CD138 and anti-B7.2, and FITC-conjugated anti-IgM andanti-CD45R/B220. All mAbs were purchased from Pharmingen (San Diego,Calif.). Briefly, Fc receptors were blocked with 10 μg/ml Fc Block(Pharmingen) for 15 min. on ice, followed by addition of PE- andFITC-conjugated mAbs and incubated on ice for 20-30 min. Cells werewashed 1× and suspended in 0.5% paraformaldehyde. Cell fluorescence datawere acquired on a FACSCalibur™ flow cytometer (Becton Dickinson, SanJose, Calif.) and analyzed using CELLQuest™ software (Becton Dickinson).

After treatment with hBAFF-R-Ig there was approximately a 50% reductionin the number of B cells in peripheral blood and in the peripherallymphoid organs examined. B220^(high) IgM^(low) B cells accounted for23.4% and 21.5% of cells in PBS-treated and HuIgG-treated mice,respectively, whereas this population represented only 9.9% of cells inhBAFF-R-Ig-treated mice. Plasma cells (sndecan/CD138+) appeared to beslightly decreased as well with 5.7% and 4.8% present in the blood ofPBS-treated and HuIgG-treated mice, respectively, compared with 3.9% inhBAFF-R-Ig-treated mice. The B7.2 molecule was upregulated on 3.1% and4.5% of B220+ cells in PBS-treated and HuIgG-treated mice, respectively,compared with 1.9% in hBAFF-R-Ig-treated mice.

In the spleen B220^(high) B cells were markedly reduced inhBAFF-R-Ig-treated mice representing 18.8%, compared with 36.7% and 40%in PBS- and HuIgG-treated mice, respectively. This decline was observedin both IgM^(high) and IgM^(low) subpopulations (see Table 8A). Therewas no change observed in the newly formed B cell compartment in thespleen, B220^(low) IgM^(high) (data not shown). Plasma cells(syndecan/CD138+) appeared to be slightly decreased as well with 3.3%and 3.4% present in the spleen of PBS-treated and HuIgG-treated mice,respectively, compared with 2.4% in hBAFF-R-Ig-treated mice.

The MLN exhibited a decline in B220+B cells with 14.1% present inhBAFF-R-Ig-treated mice compared with 26.7% and 35.8% in PBS-treated andHuIgG-treated mice, respectively. The data are summarized in Table 8A.

TABLE 8A B cell populations in hBAFF-R-Ig, PBS and HuIgG-treated mice¹.Blood B220^(high) B7.2/ Igm^(low) Syndecan B220^(low) PBS 23.4 ± 5.7 5.7± 1.5 3.1 ± 0.5 HuIgG 21.5 ± 4.5 4.8 ± 0.9 4.5 ± 1.0 HBAFF-  9.9 ± 1.83.9 ± 0.6 1.9 ± 0.5 R-Ig Spleen B220^(high) B220^(high) Igm^(low) IgM⁺Syndecan PBS 27.8 ± 1.6 11.9 ± 1.6 3.3 ± 0.8 HuIgG 30.5 ± 2   11.8 ± 1.03.4 ± 0.7 HBAFF- 10.6 ± 0.2  8.4 ± 0.2 2.4 ± 0.2 R-Ig MLN B220⁺ PBS 26.7HuIgG 35.8 ± 3.3 HBAFF-R-Ig 14.1 ± 5.9 ¹The mice were treated asdescribed in the Materials and Methods section, and the data are givenas percent ± standard

Deviation

The decreased percentage of B7.2+B cells in the blood and plasma cellsin the blood and spleens of hBAFF-R-Ig-treated mice after immunizationwith SRBCs suggests that there is inhibitation of B cell activationand/or maturation, and potentially increased elimination of activated Bcells. A very minor percent of antigen-specific B cells would beactivated and respond to any antigen, in this case SRBC. Because thehBAFF-R-Ig treatment resulted in such a dramatic reduction in thepercent of B cells in all tissues examined, ˜50%, the activity ofhBAFF-R-Ig appears to also target resting, mature B cells.

It is therefore contemplated that BAFF-R fusion protein may be used as atherapeutic drug with clinical application in B cell-mediated diseases.Diseases include those that are autoimmune in nature such as systemiclupus erythematosus, myasthenia gravis, autoimmune hemolytic anemia,idiopathic thrombocytopenia purpura, anti-phospholipid syndrome, Chaga'sdisease, Grave's disease, Wegener's Granulomatosis, Poly-arteritisNodosa and Rapidly Progressive Glomerulonephritis. The therapeutic agentalso has application in plasma cell disorders such as multiple myeloma,Waldenstrom's macroglobulinemia, Heavey-chain disease, Primary orimmunocyte-associated amyloidosis, and Monoclonal gammopathy ofundetermined significance (MGUS). Oncology targets include B cellcarcinomas, leukemias, and lymphomas.

Example 9: Blocking BAFF Induced B Cell Proliferation In Vitro UsingSoluble BAFF-R

In this example we show that the soluble BAFF-R: hIgG1 fusion protein iscapable of blocking BAFF induced B cell proliferation in mousesplenocytes.

Murine splenocytes (5×10⁵ cells/ml) from Balb/c mice were isolated andcultured in a 96-well plate in 100 μl of RPMI (Life Technologies)supplemented with 10% fetal calf serum (JRH), 2 mM glutamine, 100units/ml penicillin and 100 mg/ml streptomycin (Life Technologies). Thenvarious amounts of human BAFF, 10 μg/ml human BAFF-R: hIgG1 or human Ig,as well as 10 μg/ml of anti-mouse surface heavy chain (Jackson ImmunoResearch) were added. After 48 h in culture at 37° C., cells were pulsedfor 24 h with 1 Ci/well [methyl-³H] thymidine (Dupont NEN) and harvestedusing a Tomtec (Orange, Conn.) cell harvester. The radioactivity wasmeasured in a Betaplate liquid scintillation counter (PharmaciaBiotech).

FIG. 8 shows the results of the splenocyte Proliferation Assay. Shownare the counts per minute (CPM) incorporated into the mouse splenocytecells vs. the amount of human BAFF reagent added. The level of anti-μ orfusion protein or control hIgG is held constant at 10 μg/ml. The solidsquares represent the level of proliferation induced by BAFF alone. Thesolid circles represent the proliferation of the cells with BAFF andanti-μ. The curve with the open triangle represents the inhibitionobserved when BAFF-R: hIgG1BAFF is added with anti-μ plus BAFF-R:hIgG1.The open diamond represents the inhibition observed using control humanIg.

The addition of BAFF plus anti-μ results in the proliferation of mousesplenocytes in vitro. The level of ³H thymidine incorporated into thecells is significantly higher than the level obtained when either BAFFalone or anti-μ alone is added to the splenocytes. The treatment of thesplenocytes with BAFF alone results in ³H-thymidine incorporation onlyat very high concentrations. The addition of anti-μ alone results in noproliferation. When 10 μg/ml of control human Ig was added to thesplenocytes with increasing amounts of BAFF and 10 μg/ml anti-μ, therewas a modest decrease in the level of proliferation. In contrast, when10 μg/ml of a human BAFF-R: hIgG1 fusion protein is added to the assayunder the same conditions, the extent of proliferation is reduced to thelevel observed for the BAFF alone treatment. This indicates that theBAFF-R: hIgG1 fusion protein is capable of inhibiting the proliferationof splenocytes induced by BAFF and anti-t.

Example 10: Blocking BAFF Binding to Raji Cells Using BAFF-R: hIgG1

In this example, pre-incubation of BAFF with BAFF-R: hIgG1 fusionprotein resulting in the reduction of BAFF binding to the Raji B celllymphoma line is described.

In this FACS assay, 200 ng/ml FLAG-tagged human BAFF was pre-incubatedwith either human BAFF-R: hIgG1 or human LTβR: hIgG1 at two folddilutions ranging from g/ml to 39 ng/ml or no fusion protein for 30 min.on ice. The incubation took place in a FACS buffer containing PBSwithout Ca²⁺ or Mg²⁺ plus 10% FCS (JRH) and 0.05% sodium azide. Afterpre-incubation, the BAFF-fusion protein mixture was added to 5×10⁶ Raji(ATCC) cells/ml. This incubation took place for 30 min. on ice and thenthe cells were washed with 2 ml of FACS buffer at 4° C. In order todetect the bound BAFF, 5 μg/ml of anti-FLAG antibody M2 (Sigma) wasadded to the cells and incubated for 30 min. on ice. The cells wereagain washed as above and then incubated with a 1:100 dilution of PEconjugated donkey anti-mouse Ig (Jackson Immuno Research) for 30 min. onice. After the, cells were washed again with FACS buffer, they werefixed with 1% paraformaldehyde and read by FACS® (Becton Dickinson andCo.)

FIG. 9 shows the results of the BAFF blocking assay. Shown are the MFI(mean fluorescence intensity) readings resulting from a FACS® analysisexamining BAFF binding to Raji cells. The solid squares represent thedata when human BAFF-R: hIgG1 is pre-incubated with BAFF prior tobinding to the cells. The circles show the curve resulting from additionof a non-specific fusion protein, human LTβR: hIgG1, to BAFF. The x-axisrepresents the amount of fusion protein added to 200 ng/ml BAFF prior toincubation with the cells.

When no fusion protein is pre-incubated with human BAFF, the meanfluorescence intensity (MFI), of the sample was 80. When human LTβR:hIgG1 is pre-incubated with BAFF, even at 20 μg/ml, the detection ofBAFF binding to Raji cells is unchanged. When human BAFF-R: hIgG1 ispre-incubated with BAFF, however, there is a substantial decrease in theMFI, down to 20-25 even at 625 ng/ml fusion protein. The background MFIfor this experiment is 7. Thus, BAFF-R: hIgG1 is very effective atblocking BAFF binding to Raji B cells.

Example 11: BAFF-R-IgG Attenuates Lupus-Like Autoimmune Disorders inTransgenic Mice

This example describes the effects of BAFF-R-Ig to reduce the peripheralB-cell pool and inhibit development of splenamegally and nephritis.

Five-month-old BAFF transgenic (Tg) mice (C57BL/6) and age-matchedlittermates were used in the experiment. BAFF Tg mice manifestlymphocytic disorders and an autoimmune phenotype similar to systemiclupus erythematosus (Mackay et al., 1999). This phenotype includesincreased peripheral B cell populations including Transitional 1 (T1)and 2 (T2), mature (M), marginal zone (MZ) and CD1^(high)/IgM^(high) Bcells.

The mice received i.p. PBS, 400 mg of purified human IgG (hIg) (Sandoz,Basel, Switzerland) (3/group) or 400 μg of CHO-derived humanBAFF-R-huIgG1 (hBCMA-Ig) fusion protein (2/group) on days 0, 4, 7, 11,14, 18, 21, 25, 28, 32, 35 and were sacrificed at day 40.

At the time of sacrifice, spleen weight was measured and single cellsuspensions were prepared after lysing red blood cells in a hypotonicbuffer. Flow cytometry was performed using FITC-conjugatedanti-CD21/CD35, PE-conjugated anti-IgD and anti-CD1, cychrome-conjugatedanti-CD45R/B220 and biotin-conjugated anti-IgM. All mAbs were purchasedfrom Pharmingen (San Diego, Calif.). Briefly, Fc receptors were blockedwith 10 μg/ml Fc Block (Pharmingen) for 10 min on ice, followed byaddition of biotin-conjugated mAbs for 30 min on ice, cells were washed1×, followed by addition of FITC, PE, Cychrome-conjugated Abs,strep-avidin APC and 10 μg/ml Fc Block. Cells were incubated for 30 minon ice, washed 1× and suspended in 0.5% paraformaldehyde. Cellfluorescence data were acquired on a FACSCalibur (flow cytometer (BectonDickinson, San Jose, Calif.) and analyzed using CELLQuest (software(Becton Dickinson).

The presence of proteins in mouse urine was measured using Multistix 10SG reagent strips for urinalysis (Bayer Corp., Diagnostics Division).

Results are shown in Tables 11A and 11B below:

TABLE 11A 3 Month Splenocyte Analysis Total Cells per Spleen (×10⁶) TlT2 MZ Mature BAFFTg Mice 816E23   9.4 18  9  91 816E33 14 30 19 170816E99 14 24 19 150 Mean 13+/2.6 24+/−6 16+/5.7 140+/−41 LittermateControls 816E2   5.3   5.8   3.1  73 816E4   2.4   3.7   1.9  44 Mean3.9+/−2 4.8+/−1.5 2.4+/−1 59+/−20The spleens of the 3 month old Baff Tg mice (n=3) and age-matchedlittermates (n=2) were isolated and subjected to FACS analyses asdescribed in the Materials and Methods. The T1, T2, M and MZ cells weredefined by the expression of the following surface markers (hi: high,lo: low, int: intermediate):

T1: IgD⁻, IgM^(high), CD21^(lo)

MZ: IgD⁻, IgM^(hi), CD21

T2: IgD⁺, IgM^(hi), CD21^(hi)

M: IgD⁺, IgM^(lo), CD21^(int)

TABLE 11B 10 Month Splenocyte Analysis Total Cells per Spleen (×10⁶) TlT2 MZ Mature BAFF Tg Mice 802-39 13   100  34   630 823-3-11 7.5 43 6.6200 823-3-13 3.1 50 15   180 Mean 7.9+/−4.9 64+/−30 19+/−10 340+/−250Littermate Controls 823-3-22 0.7   6.5 1.4  56 802-64  0.28   5.4 1.3 38 823-14-13  0.45   3.4  0.16  38 Mean 0.48+/−0.21 5.1+/−1.50.95+/−0.65 44+/−1010 month old Baff Tg mice (n=3) and age-matched littermates (n=3) weresubjected to FACS analyses as described in Table 1.

After administration of h BAFF-R-Ig to Baff Tg mice the T1, T2, M, MZpopulations and the CD1^(high)/IgM^(high) B cells in the spleen werereduced significantly as compared to the PB S and hIg treated Baff Tgmice, the total numbers of T1, T2, M, MZ and CD1^(high)/IgM^(high) Bcells were reduced to the level similar or lower than that of thecontrol littermates treated with PBS (FIG. 10a, 10b, 10c ).

BAFF-R-Ig treatment of Baff Tg mice resulted in an attenuation ofBaff-mediated autoimmune disease as evidenced by the observation thatBAFF-R-treated mice had spleens of normal size while control-treatedBaff Tg mice exhibited splenamegally (FIG. 11). In addition, thedevelopment of proteinuria, an indicator of renal dysfunction, wasinhibited in BAFF-R-Ig treated mice, whereas control-treated Baff Tgmice developed nephritis as determined by rising proteinuria levels(FIG. 12).

Mice transgenic for Baff have vastly increased numbers of peripheral Bcells, and our further analysis revealed predominantly increased T1, T2,MZ B cell subpopulations in these mice. The transitional stage of B celldevelopment (T1 and T2) is the checkpoint at which autoreactive B cellsare presumably eliminated. CD1^(h)/IgM^(high) B cells, which tend toreside in the marginal zone, were also highly increased in Baff Tg mice.This latter population of B cells was shown to be the major source ofautoantibody production in NZB/NZW lupus mice. Treatment of Baff Tg micewith BAFF-R-Ig results in a reduction of the T1, T2, MZ and CD1^(hi) Bcell populations to levels similar to or lower than those found inwildtype littermate controls.

BAFF-R-Ig functioned to reduce the peripheral B cell pool and inhibitdevelopment of splenamegally and nephritis. Therefore, in addition toits usefulness in systemic lupus erythematosus lupus nephritis, theaction of BAFF-R-Ig is also useful as in B cell-mediated diseases thatare autoimmune in nature such as myasthenia gravis, autoimmune hemolyticanemia, idiopathic thrombocytopenia purpura, anti-phospholipid syndrome,Chaga's disease, Grave's disease, Wegener's Granulomatosis,Poly-arteritis Nodosa and Rapidly Progressive Glomerulonephritis. Thistherapeutic agent also has application in plasma cell disorders such asmultiple myeloma, Waldenstrom” macroglobulinemia, Heavey-chain disease,Primary or immunocyte-associated amyloidosis, and Monoclonal gammopathyof undetermine significance (MGUS). Oncology targets include B cellcarcinomas, leukemias, and lymphomas.

Example 12: Mean Arterial Pressure in BAFF Transgenic Mice

This experiment describes the measurement of blood pressure in BAFFtransgenic mice. Observations made during phenotype evaluation of BAFFtransgenic mice indicated the potential for hypertension in the mice.

Baff Tg mice as described above were anesthetized with ketamine. Theleft carotid artery was exposed via an incision in the neck. A catheterwas inserted into the carotid artery for measurement of blood pressureand heart rate. The catheter was connected to a pressure transducer andblood pressure was measured using a Gould data acquisition system(Po-Ne-Mah Data Acquisition system and Gould polygraph). From thepulsatile pressure waveform, systolic pressure, diastolic pressure, meanpressure and heart rate were derived. Two groups of mice were used: BAFFtransgenics (n=8) and Wild-Type Controls (n=9).

FIG. 13 displays the average mean arterial pressure (MAP, in mmHg) forthe two groups. As shown, the BAFF transgenic mice had an average MAP of102±8 mmHg. The control mice had an average MAP of 92±6 mmHg. Thus, theBAFF mice demonstrate a 10 mmHg increase in MAP over controls, althoughthis did not achieve statistical significance (ANOVA).

More detailed analysis of the data are shown in FIG. 14. In this figure,the results from individual animals are displayed. In the wild-typecontrols (BAFF −), the distribution of the data follow a typical,binomial distribution. In contrast, the pressures in the BAFF transgenicmice (BAFF +) appear as if they distribute into two groups, with onegroup in the 120-130 mmHg range and one group in the 80-90 mmHg range.

As a population, the BAFF transgenic mice have a tendency towardshypertension, as compared to negative control mice. Analysis ofindividual levels of arterial pressure indicate the BAFF transgenic miceare distributed into two sub-populations, one with high blood pressureand one with normal blood pressure. Accordingly, administering a solubleBAFF-R, fusion protein or antibody homolog can ameliorate the effects ofhypertension.

Example 13: BAFF-R-Ig Treatment of SNF₁ Mice with Established DiseaseSlows Progression to Severe Nephritis

Female lupus-prone (SWR x NZB)F1 (SNF₁) mice, aged 21 weeks andexhibiting moderate nephritis (30-100 mg/dl proteinuria), received 200μg of fusion protein human BAFF-R-huIgG1 (hBCMA-Ig), human IgG (HuIgG)(Sandoz), or 200 μl of PBS i.p. weekly for 8 weeks. The urine of eachmouse was monitored weekly for proteinuria using Albustix (Bayer Corp.,Terrytown, N.Y.). Proteinuria over 100 mg/dl was scored as severenephritis. BAFF-R-Ig was produced from transiently transfected EBNA 293cells. Conditioned media from 293 cells over-expressing hBCMA-Ig wasloaded onto a protein A column. Protein was eluted using 25 mM phosphate100 mM NaCl pH 2.8 following by neutralization with 1/20 volume of 0.5 MNaPO4 pH 8.6. Selected fractions based in OD280 were subjected toreducing and non-reducing SDS-PAGE gels and Western blots to identifythe purified protein.

Three weeks after treatment was terminated 50% of mice treated withBAFF-R-Ig exhibited severe nephritis, compared to 75% and 87.5% of micethat received HuIgG and PBS, respectively (see FIG. 15). These datademonstrate that the soluble BAFF receptor, BCMA-Ig, can function toblock B cell-mediated autoimmune disease, such as lupus nephritis,resulting in a marked delay in disease progression.

Example 14: BAFF-R-Ig Treatment of Normal Mice Results in a Reduction inthe Number of Splenic Dendritic Cells (DC) and an Atypical SplenicLocalization

Seven-week-old female BALB/c mice (3/group) were given i.p. either 20 μgor 50 μg human BAFF-R-IgG1 (hBCMA-Ig), 50 μg human IgG (HuIgG) or 100 μlPBS 1×/week for 4 weeks. The hBCMA-Ig fusion protein was purified fromculture supernatants of a stably transfected CHO cell line. Spleens wereobtained 8 days after the last injection and digested with collagenase(Sigma cat. # C-5138) for 1 hr. at 37° C. A single cell suspension wasprepared, RBCs were lysed in a hypotonic buffer, and the cells werewashed 3× in PBS. Splenocytes were prepared for flow cytometric analysisby staining cells with anti-CD11c-biotin followed by streptavidin-APC,anti-CD8a-cychrome, and anti-CD4-FITC to assess DC populations.

In a separate experiment female BALB/c (N=3) mice were given i.p. 100 μghBCMA-Ig 1×/week for 4 weeks after which spleens were snap frozen in OCTusing 2-methyl butane chilled on CO2. Cryosections were cut and fixed inacetone. Sections were incubated with anti-CD11c-biotin followed bystreptavidin-AP and substrate BCIP (Pierce) to visualize DCs, mAb MOMA-1followed by anti-rat IgG-HRP (Jackson ImmunoResearch) and substrate 3,3′diaminobenzidine (Sigma, St. Louis Mo., Cat. # D-1293) to visualizemetallophilic macrophages, and anti-CD35-biotin followed by streptavidinAP and substrate (Alkaline Phosphatase Substrate Kit I, Vector cat #SK-5100) to visualize follicular dendritic cells (FDC). Mice treated invivo with either 20 μg or 50 μg of BCMA-Ig 1×/week for 4 weeks exhibiteda significant decline (p<0.05 by Student's t test) in splenic CD11c+ DCsas compared to HuIgG and PBS-treated controls. This decline was seen forall CD11c+DC populations examined: CD8a−CD4−, CD8a+CD4−, andCD8a−CD4+(FIG. 16, Table 14A).

TABLE 14A BAFF-R-Ig treatment results in a reduction in the number ofsplenic DCs. PBS HuIgG 20 μg BCMA-Ig 50 μgBCMA-Ig Mean number of CD11+DCs (×10⁶) ± SD CD8a − CD4− 0.81 ± 0.15 0.49 ± 0.07 0.26 ± 0.04 0.35 ±0.05 CD8a + CD4− 0.36 ± 0.02 0.35 ± 0.07 0.16 ± 0.02  0.2 ± 0.02 CD8a −CD4+ 0.92 ± 0.02 0.84 ± 0.15  0.4 ± 0.06 0.55 ± 0.04

This decline in splenic DCs, which are critical antigen presentingcells, may impact B cell activation, maturation and humoral immunity.

Frozen spleen sections were obtained from mice as described in Materialsand Methods. BCMA-Ig-treated mice exhibited an atypical DC homingpattern when stained with anti-CD11c. DCs normally localize within the Tcell area of the white pulp and within the marginal zone, with aconcentration at the marginal zone bridging channels. However, DCs fromBCMA-Ig-treated mice were found surrounding the perimeter of themarginal zone and few appeared to be able to migrate further within thewhite pulp (data not shown). Therefore, blocking BAFF/BAFF-R interactionwith soluble BAFF receptor appears to interfere with the homing patternof DCs which may affect their ability to function as antigen presentingcells, thereby impacting B cell activation, maturation and humoralimmunity. Furthermore, the spleens of BCMA-Ig-treated mice lacked CD35+FDCs as determined by immunohistochemistry (data not shown). Since FDCsfunction to present antigen to B cells within germinal centers (GC),lack of such cells could have a detrimental effect on GC structure and Bcell affinity maturation.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the polypeptides,compositions and methods of the invention without departing from thespirit or scope of the invention. Thus, it is intended that the presentinvention cover the modifications and variations of this inventionprovided that they come within the scope of the appended claims andtheir equivalents.

1-21. (canceled)
 22. A method of treating a renal disorder in a mammal,the method comprising the step of administering to the mammal aneffective amount of a BCMA (B-Cell Maturation Antigen) polypeptide,wherein the BCMA polypeptide comprises an amino acid sequence that bindsto BAFF (B-Cell Activating Factor of the TNF family; SEQ ID NO:9) andis: a) at least 95% identical to amino acids 1-51 of SEQ ID NO:1; or b)at least 95% identical to amino acids 8-41 of SEQ ID NO:1.
 23. Themethod of claim 22, wherein the BCMA polypeptide comprises an Fc domainof an immunoglobulin.
 24. The method of claim 22, wherein the mammal ishuman.
 25. The method of claim 22, wherein the BCMA polypeptidecomprises the sequence of amino acids 1-51 of SEQ ID NO:1.
 26. Themethod of claim 22, wherein the BCMA polypeptide comprises the sequenceof amino acids 8-41 of SEQ ID NO:1.
 27. The method of claim 23, whereinthe immunoglobulin is IgG.
 28. The method of claim 23, wherein theimmunoglobulin is human.